The human body encounters most pathogens through mucosal sites. Two main portals are the lung and gut, where pathogens are encountered through the aerodigestive tract mucosa and oral ingestion, respectively. While both of these organs harbor resident immune cells for providing host defense in these tissues, this indigenous immunity is not sufficient to protect against succumbing to illness and even mortality from prevalent respiratory and intestinal pathogens such as influenza, respiratory syncytical virus (RSV), rotavirus and numerous bacterial infections that pose substantial threats to world health. For all of these mucosal pathogens, current vaccine approaches, when available, generate only systemic immune responses and none specifically target mucosal sites nor do they promote durable, immune memory at mucosal sites. What is required is a better understanding of the nature of the immune response and cellular players at mucosal sites, to design new strategies for boosting immunity for immediate destruction of pathogens at their portal of entry. A subset of T cells, designated memory T cells, are important targets for enhancing mucosal immunity because they are the predominant T cell subset that migrates to and resides in mucosal sites such as the lung and gut, and they can mediate rapid effector functions for efficacious protective immunity. In mice, memory T cells are generated following activation and clonal expansion of antigen-specific naive T cells, persist long-term in vivo, and mediate robust secondary responses upon pathogen challenge both in the lymphoid tissue and at the site of infection. In humans, studies are generally limited to peripheral blood, and suggest similar memory functions and heterogeneity;however, little is known regarding the properties of memory T cells against mucosal pathogens in various anatomic compartments. In addition, most prior studies of tissue-specific human immune responses are limited to the sampling of individual tissues that have been surgically excised due to disease. Our overall goal in this challenge application application is to move substantially beyond the current knowledge of immunity at human mucosal sites by taking a novel, "whole-body" approach to investigate the functional potential and regulation of T lymphocytes in mucosal sites, by analyzing memory T cells in healthy mucosal and lymphoid tissues obtained from individual organ donors through a novel collaboration with the Transplant Resource Center of Maryland. The central hypothesis of this study is that memory T cells in mucosal tissue sites (mucosal memory T cells) exhibit more effector-like properties, are enriched in specificities for mucosal pathogens, and have increased homeostasis compared to lymphoid memory T cells. In the proposed research, we will analyze the distribution, antigen-specificity, functional and migration capacities, and homeostasis of human memory CD4 and CD8 T cells in mucosal versus lymphoid tissue sites using tissues obtained from individual organ donors with a focus on the lung and gut as key mucosal sites. We will determine if mucosal memory T cells exhibit specialized functions upon recall, and whether they exhibit distinct homing properties compared to lymphoid memory T cells. We will also assess how antigen-specific memory T cells are distributed in mucosal versus lymphoid sites, and whether there is a biased distribution of memory T cells specific for pathogens that infect the lung or gut, in the infected tissue site. Finally, we will also analyze how mucosal versus lymphoid memory T cells are maintained and whether they differ in their homeostatic turnover and ability to respond to survival factors. The results obtained from the proposed research will fill a notable gap in our knowledge of human immune responses at mucosal sites that is crucial for designing vaccines to generate efficacious and long-lasting mucosal immune responses. Improving immune memory at mucosal sites would have widespread and global impact on the way we treat and prevent infectious disease. PUBLIC HEALTH RELEVANCE: Pathogens that infect mucosal sites including influenza, tuberculosis, respiratory syncytial virus (RSV), rotavirus, and numerous intestinal parasites and bacteria pose substantial threats to world health, and have eluded the generation of protective vaccines. A subset of T cells, designated memory T cells, are important targets for boosting immunity against these pathogens due to their ability to migrate to and reside in mucosal tissue and their rapid protective responses. In the proposed research, we will take a novel "whole-body" approach to investigate the functional potential and longevity of human mucosal memory T cells obtained from individual organ donors, with a focus on the lung and intestine as key mucosal sites. The results obtained will reveal how human mucosal memory T cells are distributed, functionally regulated and persist, paving the way for development of new strategies to improve the efficacy of vaccines and human host defense against mucosal pathogens.